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Page 12 of 36 Dave et al. J Cancer Metastasis Treat 2020;6:46 I http://dx.doi.org/10.20517/2394-4722.2020.106

increased in combination with superoxide dismutase (SOD) with any anthocyanidins bearing the ortho-
dihydroxyphenyl arrangement on the B-ring. Multiplicative analysis demonstrated that inhibition between
delphinidin and SOD is synergistic in nature [139] . Therefore, even though the signaling pathways affected by
SOD or delphinidin are different, both are believed to play a crucial role in the cancer preventive activity of
anthocyanidins.

Suppression of inflammation through NF-κB and COX-2
There are some antioxidants that block the expression of COX-2 by inhibiting the signaling mechanism that
controls the COX-2 gene [143] . In one study, the molecular mechanism of anthocyanins was evaluated using
the mouse macrophage cell line RAW264. Bilberry and purified delphinidin derived anthocyanin extracts
suppress LPS-induced COX-2 expression at the transcription and protein levels. Signal pathway analysis
showed that delphinidin blocks LPS-induced IκB degradation and inhibits NF-κB stimulation and COX-2
gene expression. Thus, it appears anthocyanins act through the NF-κB signaling pathway which is involved
in the suppression of COX-2 gene expression [144] .

Apoptotic induction of cancer cells (by targeting ROS and JNK mediated caspase activation)
Petunidin, delphinidin, and cyanidin induced apoptosis with HL-60 cells, as demonstrated by DNA
fragmentation and morphological changes, although peonidin, malvidin, and pelargonidin did not induce
this response [145] . The number of hydroxyl groups at the B-ring is proportional to the potency of apoptosis
induced by anthocyanidins; the ortho-dihydroxyphenyl structure at the B-ring is vital for apoptosis
induction [145] .

Mechanistic analysis indicated the induction of apoptosis by delphinidin may involve an oxidation/
JNK-mediated caspase pathway. Delphinidin increases intracellular ROS which may trigger JNK. With
delphinidin treated cells, JNK phosphorylation, caspase-3 activation, and c-jun gene expression were
observed [145] . Delphinidin-induced JNK phosphorylation, DNA fragmentation, and caspase-3 activation
were effectively blocked by antioxidants such as N-acetyl-L-cysteine [145] . Thus, delphinidin can potentiate
the apoptotic death program in HL-60 cells through JNK signaling mediated through oxidative stress.

Chemopreventive mechanisms of anthocyanins
Anti-carcinogenic activities at the initial stage of tumorigenesis
Anthocyanins can prevent the occurrence of tumors by acting on antioxidant systems [146] . These
compounds scavenge free radicals reducing damage to the genome of the normal cells by oxidative stress
and later malignant transformation fostered by gene mutations [147,148] . Anthocyanins can induce the
antioxidant effect by acting on the antioxidant response element through KEAP1-Nrf2. Furthermore,
by regulating the expression of phase II antioxidases (quinone oxidoreductase, glutathione transferase,
glutathione peroxidase, and glutathione reductase), anthocyanins can suppress the activity of caspase-3.
The abnormal secretion and overexpression of inflammatory elements are important for tumorigenesis. It
has been reported that anthocyanins can mediate anti-inflammatory functions by regulating the secretion
and expression of inflammatory agents. This effect occurs due to the suppression of transcription factors
NF-κB through multiple pathways [149,150] . In an in vitro study, anthocyanin containing purple-fleshed potato
extracts elevated apoptosis and suppressed proliferation in a p53-independent manner in colon CSCs [151] .
In vivo, purple-fleshed potato decreased the number of crypts containing cells with nuclear β-catenin
(which is an indicator of colon CSCs) by induction of apoptosis [151] . These results provide evidence that
anthocyanins may reduce the initial stage of tumorigenesis.

Anti-carcinogenic activities in the cancer formation stage
Anthocyanins can block tumorigenesis and induce terminal differentiation of tumor cells. It was found that
differentiation of the acute promyelocytic leukemia cell line HL-60 could be induced by cyanidin-3-O-β-